Noncontiguous finished genome sequence and description of Nocardioides massiliensis sp. nov. GD13(T).

Nocardioides massiliensis sp. nov strain GD13(T) is the type strain of N. massiliensis sp. nov., a new species within the genus Nocardioides. This strain was isolated from the faeces of a 62-year-old man admitted to intensive care for Guillain-Barré syndrome. Nocardioides massiliensis is a strictly aerobic Gram-positive rod. Herein we describe the features of this bacterium, together with the complete genome sequence and annotation. The 4 006 620 bp long genome contains 4132 protein-coding and 47 RNA genes.

Introduction

Nocardioides massiliensis strain GD13T (= DSM 28216 = CSUR P894) is the type strain of N. massiliensis sp. nov. This bacterium was isolated from the human faeces of a 62-year-old man admitted to intensive care for a Guillain-Barré syndrome as part of an effort called culturomics to cultivate all bacterial species from the human gut [1], [2]. The current taxonomic classification of prokaryotes is based on a combination of phenotypic and genotypic characteristics [3], [4], which includes 16S rRNA gene phylogeny, G+C content and DNA-DNA hybridization. Although these tools are considered the reference standard, they have several drawbacks [5], [6]. As a result of the declining cost of sequencing, the number of sequenced bacterial genomes has rapidly grown; to date, almost 40 000 bacterial genomes have been sequenced (GOLD Database, https://gold.jgi.doe.gov/). Hence, we recently proposed to use genomic information together with phenotypic criteria for the description of new bacterial species [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20].

Here we present a summary classification and a set of features for Nocardioides massiliensis sp. nov. strain GD13T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species Nocardioides massiliensis. The genus Nocardioides was created in 1976 (Prauser, 1976). The first two species were Nocardioides albus and Nocardioides luteus [21], [22]. These Gram-positive, aerobic and cardioform actinomycetes exhibit a high G+C content and are able to develop mycelium [21], [22]. Currently the genus Nocardioides contains 72 species, including eight described in 2013 and species from the genera Pimelobacter and Arthrobacter that were reclassified in the genus Nocardioides [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36] (LPSN, http://www.bacterio.net/). Members of the genus Nocardioides are mostly environmental bacteria found in soil or plants. To our knowledge, this is the first report of isolation of a Nocardioides species from humans.

Organism Information and Classification

A stool sample was collected from a 62-year-old man admitted to the intensive care unit of Hôpital Nord, Marseille, France, in March 2012 [2]. The study was approved by the ethics committee of the Institut Fédératif de Recherche IFR48 (Faculty of Medicine, Marseille, France), under agreement 09-002. At the time of sampling, the patient had received a 10-day course of imipenem (3 g per day). The faecal specimen was preserved at −80°C after collection. Strain GD13T (Table 1) was isolated in March 2012 by cultivation on PVX agar (bioMérieux, Marcy l’Étoile, France) in an aerobic atmosphere at 28°C after 14 days of incubation. Strain GD13T exhibited a 98.07% 16S rRNA sequence identity with N. mesophilus (GenBank accession no. EF466117), the phylogenetically closest bacterial species with a validly published name (Figure 1). Its 16S rRNA sequence was deposited in European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL-EBI) under number HF952922. This value was lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers [4] to delineate a new species without carrying out DNA-DNA hybridization.

Table 1

Classification and general features of Nocardioides massiliensis strain GD13T according to MIGS recommendations

MIGS ID	Property	Term	Evidence codea
	Current classification	Domain: Bacteria	TAS [37]
		Phylum: Actinobacteria	TAS [38]
		Class: Actinobacteria	TAS [39]
		Order: Actinomycetales	TAS [39], [40], [41], [42]
		Family: Nocardioidaceae	TAS [39], [42], [43], [44]
		Genus: Nocardioides	TAS [21]
		Species: Nocardioides massiliensis	IDA
		Type strain GD13T	IDA
	Gram stain	Positive	IDA
	Cell shape	Rod shaped	IDA
	Motility	Not motile	IDA
	Sporulation	Nonsporulating	IDA
	Temperature range	Mesophile	IDA
	Optimum temperature	37°C	IDA
MIGS-6.3	Salinity	Unknown	IDA
MIGS-22	Oxygen requirement	Aerobic	IDA
	Carbon source	Unknown	IDA
	Energy source	Unknown	IDA
MIGS-6	Habitat	Human gut	IDA
MIGS-15	Biotic relationship	Free-living	IDA
MIGS-14	Pathogenicity	Unknown
	Biosafety level	2
	Isolation	Human faeces
MIGS-4	Geographic location	Marseille	IDA
MIGS-5	Sample collection time	March 2012	IDA
MIGS-4.1	Latitude	43.296482	IDA
MIGS-4.1	Longitude	5.36978	IDA
MIGS-4.3	Depth	Surface	IDA
MIGS-4.4	Altitude	0 m above sea level	IDA

MIGS, minimum information about a genome sequence.

Evidence codes are as follows: IDA, inferred from direct assay; and TAS, traceable author statement (i.e. a direct report exists in the literature). These evidence codes are from the Gene Ontology project (http://www.geneontology.org/GO.evidence.shtml) [45]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or by an expert or reputable institution mentioned in the acknowledgements.

Fig. 1

Phylogenetic tree highlighting position of Nocardioides massiliensis strain GD13T relative to other type strains within Nocardioides genus. Strains and their corresponding GenBank accession numbers for 16S rRNA genes are (type = T): Nocardioides massiliensis strain GD13T, HF952922; N. mesophilus strain MSL-22T, EF466117; N. iriomotensis strain IR27-S3T, AB544079; N. jensenii strain ATCC 49810T, AF005006; N. daedukensis strain MDN22T, FJ842646; N. daejeonensis strain MJ31T, JF937066; N. dubius strain KSL-104T, AY928902; N. halotolerans strain DSM 19273T, EF466122; N. daphniae strain D287T, AM398438; N. salarius strain CL-Z59T, DQ401092; N. basaltis strain J112T, EU143365; N. ginsengisegetis strain Gsoil 48T, GQ339901; N. kribbensis strain KSL-2T, AY835924; N. alkalitolerans strain DSM 16699T, AY633969; N. insulae strain DSM 17944T, DQ786794; Aeromicrobium massiliense strain JC14T, JF824798. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum-likelihood method within MEGA6. Numbers at nodes are percentages of bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree. Only bootstrap values equal to or greater than 70% are indicated. Aeromicrobium massiliense was used as outgroup. Scale bar = 1% nucleotide sequence divergence.

Phenotypic and biochemical features

Growth was tested at different temperatures (25, 30, 37, 45, 56°C). It occurred between 25 and 37°C after 24 to 48 hours of incubation. No growth was observed at 45 or 56°C. Colonies were 0.5 mm in diameter on 5% sheep's blood–enriched Columbia agar (bioMérieux). Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems (bioMérieux) respectively, and under aerobic conditions, with or without 5% CO2. It was achieved only aerobically. Gram staining showed Gram-positive, rod-shaped bacteria unable to form spores (Figure 2). A motility test was negative. Cells grown on agar were soft and yellowish in colour after 24 to 48 hours and could be grouped in small clumps (Figure 2). They had a mean width of 0.46 μm and a mean length of 1 μm (Figure 3).

Fig. 2

Gram staining of Nocardioides massiliensis strain GD13T.

Fig. 3

Transmission electron microscopy of Nocardioides massiliensis strain GD13T, using Morgani 268D (Philips, Amsterdam, The Netherlands) at operating voltage of 60 kV. Scale bar = 500 nm.

Strain GD13T exhibits catalase activity, but was negative for oxidase. Using an API ZYM strip (bioMérieux), positive reactions were observed for esterase (C4), esterase lipase (C8), acid phosphatase, alkaline phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, α-glucosidase and β-glucosidase; negative reactions were observed for β-galactosidase, α-galactosidase, valine arylamidase, cystine arylamidase, β-glucuronidase, lipase (C14), trypsin, α-chymotrypsin, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using rapid ID32A (bioMérieux), positive reactions were observed for arginine arylamidase, proline arylamidase and α-fucosidase and negative reactions were observed for arginine dihydrolase and N-acetyl-β-glucosaminidase. Using rapid ID32A (bioMérieux), positive reactions were observed for arginine arylamidase, proline arylamidase and α-fucosidase; negative reactions were observed for nitrate reduction, urease, production of indole, arginine dihydrolase, N-acetyl-β-glucosaminidase, serine arylamidase, histidin arylamidase, phenylalanine arylamidase and tyrosin arylamidase. Using an API 50 CH strip (bioMérieux), positive reactions were recorded for esculin hydrolysis and fermentation of d-lactose; negative reactions were obtained for fermentation of glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, l-xylose, d-adonitol, methyl-βd-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, methyl-αd-xylopyranoside, methyl-αd-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, d-cellobiose, d-maltose, d-mellibiose, d-trehalose, inulin, d-melezitose, d-raffinose, amidon, glycogen, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, l-arabitol, d-sorbitol, d-saccharose, xylitol, d-arabitol, potassium-5-ketogluconate and potassium 2-ketogluconate. Cells were susceptible to penicillin G, amoxicillin, amoxicillin/clavulanic acid, ceftriaxone, imipenem, vancomycin, rifampicin, erythromycin, gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole and resistant to metronidazole. Compared to N. mesophilus, its phylogenetically closest neighbor, N. massiliensis differed in α-glucosidase, β-glucosidase, esterase–lipase production and utilization of d-glucose and d-mannose (Table 2). In addition, Nocardioides species clearly differed from Aeromicrobium massiliense, a representative species from another genus within the family Nocardioidaceae.

Table 2

Differential characteristics of Nocardioides massiliensis strain GD13T (data from this study) compared to N. dubius strain KSL-104T; N. mesophilus strain MSL-22T; N. iriomotensis strain IR27-S3T; N. daedukensis strain MDN22T, Aeromicrobium massiliense strain JC14T

Property	N. massiliensis	N. dubius	N. mesophilus	N. iriomotensis	N. daedukensis	A. massiliense
Cell diameter (μm)	0.46 × 1	0.8–1.06 × 1.5–2.5	0.3–0.8 × 60.9–1.4	0.4–0.5 × 0.4–1	0.4–0.86 × 0 .8–3.0	1 × 1.7
Oxygen requirement	Aerobic	Aerobic	Aerobic	Aerobic	Aerobic	Aerobic
Gram stain	+	+	+	+	+	+
Motility	−	+	−	−	−	+
Endospore formation	−	−	−	−	+	−
Production of:
Alkaline phosphatase	+	+	+	+	−	−
Acid phosphatase	+	+	+	+	−	−
Catalase	+	+	+	+	+	+
Oxidase	−	+	−	NA	+	−
Nitrate reductase	−	−	−	+	+	+
Urease	−	NA	−	NA	NA	−
α-Galactosidase	−	−	−	−	−	−
β-Galactosidase	−	−	−	+	−	+
β-Glucuronidase	−	−	−	−	−	w
α-Glucosidase	+	+	−	+	−	+
β-Glucosidase	+	+	−	+	−	+
Esterase	+	−	+	+	−	+
Esterase lipase	+	−	−	+		+
Naphthol-AS-BI-phosphohydrolase	+	NA	+	+	w	−
N-acetyl-β-glucosaminidase	−	−	−	−	−	−
α-Mannosidase	−	−	−	−	−	−
α-Fucosidase	−	−	−	−	−	−
Leucine arylamidase	+	+	+	+	+	+
Valine arylamidase	−	−	−	−	−	−
Cystine arylamidase	−	−	−	+	−	−
α-Chymotrypsin	−	−	−	−	−	−
Trypsin	−	−	−	−	−	+
Utilization of:
5-Keto-gluconate	−	NA	NA	NA	NA	+
d-Xylose	−	−	+	NA	−	−
d-Fructose	−	−	−	NA	−	−
d-Glucose	−	−	+	NA	−	+
d-Mannose	−	−	+	NA	−	−
Habitat	Human gut	Soil	Soil	Soil	Soil	Human gut

+, positive result; −, negative result; w, weakly positive result; NA, data not available.

MALDI-TOF analysis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) protein analysis was carried out as previously described [46]. Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate and spread it as a thin film on a MSP 96 MALDI-TOF target plate (Bruker Daltonics, Leipzig, Germany). Twenty distinct deposits from 20 isolated colonies were performed for strain GD13T. Each smear was overlaid with 2 μL of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 2.5% trifluoracetic acid) and allowed to dry for 5 minutes. Measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2000 to 20 000 Da (parameter settings: ion source 1 (ISI), 20 kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 240 shots with variable laser power. The time of acquisition was between 30 seconds and 1 minute per spot. The 20 GD13T spectra were imported into MALDI BioTyper 3.0 software (Bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra of 7379 bacteria, including 48 spectra from two Nocardioides species. It corresponded to the Bruker database combined with our own database constantly incremented with new species. The method of identification included the m/z from 3000 to 15 000 Da. For every spectrum, a maximum of 100 peaks were compared with spectra in the database. The resulting score enabled the identification or not of tested species: a score of ≥2 with a validly published species enabled identification at the species level, a score of ≥1.7 but <2 enabled identification at the genus level and a score of <1.7 did not enable any identification. No significant MALDI-TOF score was obtained for strain GD13T against the Bruker database and against our own database, suggesting that our isolate was not a member of a known species. We added the spectrum from strain GD13T to our database (Figure 4). Finally, the gel view showed the spectral differences with other members of the genus Nocardioides and Aeromicrobium massiliense, a member of the genus Aeromicrobium that is closely related to the genus Nocardioides (Figure 5).

Fig. 4

Reference mass spectrum from Nocardioides massiliensis strain GD13T. Spectra from 17 individual colonies were compared and reference spectrum was generated.

Fig. 5

Gel view comparing Nocardioides massiliensis strain GD13T. Gel view displays raw spectra of loaded spectrum files arranged in pseudo-gel-like look. x-axis records m/z value. Left y-axis displays running spectrum number originating from subsequent spectra loading. Peak intensity is expressed in arbitrary units by greyscale scheme code, as indicated on right y-axis. Displayed species are indicated at left.

Genome Sequencing Information

Genome project history

Nocardioides massiliensis GD13T was selected for sequencing on the basis of its phenotypic differences, phylogenetic position and 16S rRNA sequence similarity to other members of the genus Nocardioides. It was part of a culturomics study aimed at isolating all bacterial species from the human digestive flora from patients treated with broad-spectrum antibiotics. It is the first sequenced genome of N. massiliensis sp. nov. The GenBank Bioproject number is PRJEB1962 and consists of 839 large contigs. Table 3 shows the project information and its association with minimum information about a genome sequence (MIGS) 2.0 compliance [47].

Table 3

Project information

MIGS ID	Property	Term
MIGS-31	Finishing quality	High-quality draft
MIGS-28	Libraries used	Mate pair
MIGS-29	Sequencing platform	MiSeq
MIGS-31.2	Fold coverage	86×
MIGS-30	Assemblers	CLCGENOMICSWB4
MIGS-32	Gene calling method	Prodigal
	EMBL-EBI Date of Release	10 September 2014
	NCBI project ID	PRJEB1962
	EMBL-EBI accession	CCXJ00000000
MIGS-13	Project relevance	Study of human gut microbiota

EMBL-EBI, European Molecular Biology Laboratory–European Bioinformatics Institute; MIGS, minimum information about a genome sequence.

Growth conditions and DNA isolation

Nocardioides massiliensis strain GD13T (= DSM 28216 = CSUR P894) was cultured aerobically on 5% sheep's blood–enriched Columbia agar (bioMérieux). Bacteria grown on four petri dishes were resuspended in sterile water and centrifuged at 4°C at 2000 × g for 20 minutes. Cell pellets were resuspended in 1 mL Tris/EDTA/NaCl (10 mM Tris/HCl (pH 7.0), 10 mM EDTA (pH 8.0), 300 mM NaCl) and recentrifuged under the same conditions. The pellets were then resuspended in 200 μL Tris-EDTA (TE) buffer and proteinase K and kept overnight at 37°C for cell lysis. DNA was purified with phenol/chloroform/isoamylalcohol (25:24:1), followed by an overnight precipitation with ethanol at −20°C. The DNA was resuspended in 205 μL TE buffer. DNA concentration was 401.63 ng/μL as measured by Qubit fluorometer using the high-sensitivity kit (Life Technologies, Carlsbad, CA, USA).

Genome sequencing and assembly

Genomic DNA (gDNA) of Nocardioides massiliensis was sequenced on a MiSeq sequencer (Illumina, San Diego, CA, USA) with two applications: paired end and mate pair. The paired end and the mate pair strategies were barcoded in order to be mixed respectively with 14 other genomic projects constructed according the Nextera XT library kit (Illumina) and 11 other projects with the Nextera Mate pair kit (Illumina).

The gDNA was diluted such that 1 ng of each strain's gDNA was used to construct the paired-end library. The tagmentation step fragmented and tagged the DNA. Then limited-cycle PCR amplification completed the tag adapters and introduced dual-index barcodes. After purification on Ampure beads (Life Technologies), the libraries were normalized on specific beads according to the Nextera XT protocol (Illumina). Normalized libraries are pooled into a single library for sequencing on the MiSeq. The pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and paired-end sequencing with dual index reads was performed in a single 39-hour run at a 2 × 250 bp read length. Within this pooled run, the index representation was determined to be 3.93%. Total information of 3.27 Gb was obtained from a 374K/mm2 density with 94.2% (6 719 000 clusters) of the clusters passing quality control (QC) filters. From the genome sequencing process, the 248 710 produced Illumina paired end reads for Nocardioides massiliensis were filtered according to the read qualities.

The mate pair library was constructed from 1 μg of gDNA using the Nextera Mate Pair Illumina guide. The gDNA sample was simultaneously fragmented and tagged with a mate pair junction adapter. The profile of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA7500 lab chip. The DNA fragments ranged in size from 1 to 8.4 kb with a mean size of 3.7 kb. No size selection was performed, and 240 ng tagmented fragments were circularized. The larger circularized DNA molecules were physically sheared to smaller sized fragments with a mean size of 839 bp on the Covaris device S2 in microtubes (Woburn, MA, USA). The library's profile and the quantitation were visualized on a High Sensitivity Bioanalyzer LabChip. The libraries were normalized to 2 nM and pooled. After a denaturation step and dilution at 10 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run was performed in a single 39-hour run at a 2 × 250 bp read length. Total mate paired run information of 3.2 Gb was obtained from a 690K/mm2 density with 95.4% (13 264 000 clusters) of the clusters passing QC filters. Within this pooled run, the index representation for Nocardioides massiliensis was determined to be 8.12%. From this genome sequencing process, the 1 027 586 produced Illumina paired end reads of N. massiliensis were filtered according to the read qualities. The reads obtained from both applications were trimmed and the optimal assembly was obtained through the CLC genomics wb4 software with 839 contigs which generated a genome size of 4.00 Mb.

Genome annotation and comparison

Open reading frames (ORFs) were predicted using Prodigal [48] with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank database [49] and the Clusters of Orthologous Groups (COGs) database using BLASTP. The tRNAScanSE tool [50] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [51] and BLASTN against the GenBank database. Lipoprotein signal peptides and the number of transmembrane helices were predicted using SignalP [52] and TMHMM [53] respectively. ORFans were identified if their BLASTP E value was lower than 1e-03 for alignment length greater than 80 amino acids. If alignment lengths were smaller than 80 amino acids, we used an E value of 1e-05. Such parameter thresholds have already been used in previous works to define ORFans.

For the genomic comparison, we compared the genome sequence of N. massiliensis strain GD13T (GenBank accession no. CCXJ00000000) with those of Nocardioides alkalitolerans strain DSM 16699 (GenBank accession no. AUFN00000000), Nocardioides halotolerans strain DSM 19273 (AUGT00000000), Nocardioides insulae strain DSM 17944 (AUGU00000000) and Aeromicrobium massiliense strain JC14T (CAHG00000000). To estimate the nucleotide sequence similarity at the genome level between N. massiliensis and other members of the family Nocardioidaceae, we determined the average genomic identity of orthologous gene sequences (AGIOS) parameter using the MAGi (Marseille Average genomic identity) software as follows: orthologous proteins were detected using the Proteinortho software (version 1.4) [54] using the following parameters: E value 1e-5, 30% percentage of identity, 50% coverage and algebraic connectivity of 50%; after fetching the corresponding nucleotide sequences of orthologous proteins for each pair of genomes, we determined the mean percentage of nucleotide sequence identity using the Needleman-Wunsch global alignment algorithm; Artemis [55] was used for data management, and DNAPlotter [56] was used for visualization of genomic features. The Mauve alignment tool was used for multiple genomic sequence alignment and visualization [57].

Genome properties

The genome of N. massiliensis strain GD13T is 4 006 620 bp long with a 71.0% G+C content (Table 4, Figure 6). Of the 4179 predicted genes, 4132 were protein-coding genes and 47 were RNAs. Three rRNA genes (one 16S rRNA, one 23S rRNA and one 5S rRNA) and 44 predicted tRNA genes were identified in the genome. A total of 2783 genes (66.59%) were assigned a putative function, and 382 genes (9.24%) were identified as ORFans. The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 4. The distribution of genes into COGs functional categories is presented in Table 5.

Table 4

Nucleotide content and gene count levels the genome

Attribute	Value	% of totala
Size (bp)	4 006 620	100
G+C content (bp)	2 844 700	71.0
Coding region (bp)	3 668 187	91.55
Total genes	4179	100
RNA genes	47	1.12
Protein-coding genes	4132	98.87
Genes with function prediction	2408	57.62
Genes assigned to COGs	2783	66.59
Genes with peptide signals	262	6.26
Genes with transmembrane helices	899	21.51

COGS, Clusters of Orthologous Groups database.

Total is based on either size of genome in base pairs or total number of protein-coding genes in annotated genome.

Fig. 6

Graphical circular map of chromosome of Nocardioides massiliensis strain GD13T. From outside in, outer two circles show ORFs oriented in forward (coloured by COGs categories) and reverse (coloured by COGs categories) direction respectively. Third circle marks rRNA gene operon (red) and tRNA genes (green). Fourth circle shows G+C% content plot. Innermost circle shows GC skew, with purple indicating negative values and olive positive values.

Table 5

Number of genes associated with 25 general COGs functional categories

Code	Value	%	Description
J	166	4.01	Translation
A	1	0.02	RNA processing and modification
K	240	5.80	Transcription
L	214	5.17	Replication, recombination and repair
B	1	0.02	Chromatin structure and dynamics
D	28	0.67	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	49	1.18	Defense mechanisms
T	112	2.71	Signal transduction mechanisms
M	130	3.14	Cell wall/membrane biogenesis
N	28	0.67	Cell motility
Z	0	0	Cytoskeleton
W	0	0	Extracellular structures
U	43	1.04	Intracellular trafficking and secretion
O	95	2.29	Posttranslational modification, protein turnover, chaperones
C	229	5.54	Energy production and conversion
G	159	3.84	Carbohydrate transport and metabolism
E	315	7.62	Amino acid transport and metabolism
F	73	1.76	Nucleotide transport and metabolism
H	126	3.04	Coenzyme transport and metabolism
I	268	6.48	Lipid transport and metabolism
P	213	5.15	Inorganic ion transport and metabolism
Q	178	4.30	Secondary metabolites biosynthesis, transport and catabolism
R	460	11.13	General function prediction only
S	190	4.59	Function unknown
—	1396	33.4	Not in COGs

COGS, Clusters of Orthologous Groups database.

Extended insights from the genome sequence

The draft genome of N. massiliensis has a larger size than that of A. massiliense (4.00 and 3.32 Mb respectively) but smaller than N. alkalitolerans, N. halotolerans and N. insulae (4.85, 4.86 and 4.06 Mb respectively). The G+C content of N. massiliensis is higher than those of N. insulae (71.0 and 70.8% respectively) but less than that of A. massiliense, N. alkalitolerans and N. halotolerans (72.6, 73.1 and 72% respectively). The gene content of N. massiliensis is larger than those of A. massiliense and N. insulae (4179, 3328 and 3702 respectively) and smaller than those of N. alkalitolerans and N. halotolerans (4634 and 4633 respectively). However, the distribution of genes into COGs categories was similar in all compared genomes (Figure 7). In addition, N. massiliensis shared 1447, 1745, 1693 and 1598 orthologous genes with A. massiliense, N. alkalitolerans, N. halotolerans and N. insulae. Among Nocardioides species, AGIOS values ranged from 75.67% between N. alkalitolerans and N. insulae to 77.56% between N. alkalitolerans and N. halotolerans. When considering N. massiliensis, AGIOS values were 75.06, 76.67 and 76.82 with N. insulae, N. alkalitolerans and N. halotolerans respectively, in the same range as among other Nocardioides species (Table 6).

Fig. 7

Distribution of functional classes of predicted genes on chromosomes of Nocardioides massiliensis (NM), Aeromicrobium massiliense (AE), Nocardioides alkalitolerans (NA), Nocardioides halotolerans (NH) and Nocardioides insulae (NI) according to COGs category. For each genome, percentage of each gene category is indicated.

Table 6

Pairwise comparison of Nocardioides massiliensis strain GD13T with other species showing (upper right) numbers of orthologous proteins shared between compared genomes and (lower left) AGIOS values

	Nocardioides massiliensis	Aeromicrobium massiliense	Nocardioides alkalitolerans	Nocardioides halotolerans	Nocardioides insulae
N. massiliensis	4132a	1447	1745	1693	1598
A. massiliense	75.17	2297a	1605	1501	1517
N. alkalitolerans	76.67	75.61	4585a	1872	1822
N. halotolerans	76.82	75.14	77.56	4677a	1753
N. insulae	75.06	74.12	75.67	76.73	3648a

AGIOS, average genomic identity of orthologous gene sequences.

Numbers of proteins per genome.

Conclusion

On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Nocardioides massiliensis sp. nov. The strain was isolated from a stool sample from a 62-year-old man admitted to intensive care in Marseille, France, for Guillain-Barré syndrome and who had received 10 days of broad-spectrum antibiotics.

Description of Nocardioides massiliensis strain GD13T sp. nov

Nocardioides massiliensis (mas.si.li.en'sis, L. masc. adj., from massiliensis of Massilia, the ancient Roman name for Marseille, France, where the type strain was isolated).

Cells are Gram-positive, nonsporulating, nonmotile and rod-shaped bacilli with a mean diameter and length of 0.46 and 1.0 μm respectively. Colonies are 0.5 mm in diameter and are smooth and pale yellow on 5% sheep's blood–enriched Columbia agar (bioMérieux). Growth occurred aerobically between 25 and 37°C.

Cells exhibit catalase activity but are oxidase negative. Positive reactions are observed for esterase (C4), esterase lipase (C8), acid phosphatase, alkaline phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, β-glucosidase, arginine arylamidase, proline arylamidase, esculin hydrolysis and fermentation of d-lactose. Negative reactions are observed for β-galactosidase, α-galactosidase, valine arylamidase, cystine arylamidase, β-glucuronidase, lipase (C14), trypsin, α-chymotrypsin, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase, arginine dihydrolase, N acetyl-β-glucosaminidase, nitrate reductase, urease, indole production, arginine dihydrolase, N acetyl-β-glucosaminidase, serine arylamidase, histidin arylamidase, phenylalanine arylamidase, tyrosin arylamidase, fermentation of glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, l-xylose, d-adonitol, methyl-βd-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, methyl-αd-xylopyranoside, methyl-αd-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, d-cellobiose, d-maltose, d-mellibiose, d-trehalose, inulin, d-melezitose, d-raffinose, amidon, glycogen, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, l-arabitol, d-sorbitol, d-saccharose, xylitol, d-arabitol, potassium-5-ketogluconate and potassium 2-ketogluconate. Cells are susceptible to penicillin G, amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, imipenem, vancomycin, rifampicin, erythromycin, gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole and resistant to metronidazole.

The G+C content of the genome is 71.0%. The 16S rRNA and whole-genome sequence of N. massiliensis strain GD13T are deposited in EMBL-EBI under the accession numbers HF952922 and CCXJ00000000 respectively. The type strain GD13T (= CSUR P894 = DSM 28216) was isolated from the faecal flora of a 62-year-old man admitted to intensive care in Marseille, France, for Guillain-Barré syndrome who had received 10 days of broad-spectrum antibiotics.